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The role of ATP and purinoceptor in osteoblast-like cell proliferation induced by mechanical stimulation

ATP和嘌呤受体在机械刺激诱导的成骨样细胞增殖中的作用

基本信息

批准号：
11671469
负责人：
NARUSAWA Ken'ichiro
金额：
$ 2.3万
依托单位：
University of Occupational and Environmental Health
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671469/
关键词：
ATP Purinoceptor MAP kinase cell proliferation MG-63 cell Mechanical stress P2X receptor osteoblast-like cell P2Y受容体 PDGF IGF-1

项目摘要

In human osteoblast-like MG-63 cells, increase of [^3H]thymidine incorporation induced by mechanical compression was inhibited by the P2 purinoceptor antagonist suramin, suggesting that mechanical stress-induced DNA synthesis was partially mediated by P2 receptors, however increase of ATP concentration after compression was not detected in supernatant fluid by our assay. Extracellular ATP increased [^3H]thymidine incorporation and cell proliferation, and synergistically enhanced the platelet-derived growth factor (PDGF)- or insulin-like growth factor-1 (IGF-1)-induced [^3H]thymidine incorporation. ATP-induced [^3H]thymidine incorporation was mimicked by the non-hydrolyzable ATPgS, and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. A P2Y receptor agonist UTP and a P2Y receptor antagonist reactive blue 2 did not affect the [^3H]thymidine incorporation, while the P2Y receptor antagonist PPADS inhibited ATP-induced [^3H]thymidine incorp … More oration, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X_4, P2X_5 and P2X_7 but not P2X_1 and P2X_3 receptors. In fura-2-loaded cells, not only ATP but also UTP increased intracellular Ca^<2+> concentration ([Ca^<2+>]i), and inhibitors for several Ca^<2+>-activated protein kinases had no effect on the ATP-induced DNA synthesis, indicating that a [Ca^<2+>]i increase is not indispensable for the ATP-induced DNA synthesis. ATP increased MAP kinase actively in a Ca^<2+>-independent manner, and synergistically enhanced the PDGF- or IGF-1-induced kinase activity. Further, the MAP kinase kinase inhibitor PD98059 abolished the ATP-induced DNA synthesis.We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors, through P2X receptors by activation MAP kinase in a Ca^<2+>-independent manner, and ATP and purinoceptor could play a important role in osteoblast-like cell proliferation induced by mechanical stimulation. Less

在人成骨细胞样 MG-63 细胞中，机械压缩诱导的 [^3H] 胸苷掺入增加被 P2 嘌呤受体拮抗剂苏拉明抑制，表明机械应力诱导的 DNA 合成部分由 P2 受体介导，但我们的测定在上清液中未检测到压缩后 ATP 浓度的增加。细胞外 ATP 增加 [^3H] 胸苷掺入和细胞增殖，并协同增强血小板源性生长因子 (PDGF) 或胰岛素样生长因子 1 (IGF-1) 诱导的 [^3H] 胸苷掺入。 ATP 诱导的[^3H]胸苷掺入被不可水解的 ATPgS 模拟，并被 P2 嘌呤受体拮抗剂苏拉明抑制，表明 P2 嘌呤受体参与其中。 P2Y 受体激动剂 UTP 和 P2Y 受体拮抗剂活性蓝 2 不影响 [^3H] 胸苷掺入，而 P2Y 受体拮抗剂 PPADS 抑制 ATP 诱导的 [^3H] 胸苷掺入，表明 ATP 诱导的 DNA 合成是由 P2X 受体介导的。 RT-PCR分析显示MG-63细胞表达P2X_4、P2X_5和P2X_7受体，但不表达P2X_1和P2X_3受体。在fura-2负载细胞中，不仅ATP而且UTP都增加了细胞内Ca^2+浓度([Ca^2+]i)，并且几种Ca^2+激活蛋白激酶的抑制剂对ATP诱导的DNA合成没有影响，表明[Ca^2+]i的增加对于ATP诱导的DNA合成并不是必不可少的。 ATP以不依赖Ca 2+ 的方式主动增加MAP激酶，并协同增强PDGF或IGF-1诱导的激酶活性。此外，MAP激酶激酶抑制剂PD98059消除了ATP诱导的DNA合成。我们得出结论，ATP通过P2X受体以不依赖Ca^2+的方式激活MAP激酶，增加DNA合成并增强生长因子的增殖作用，并且ATP和嘌呤受体在机械诱导的成骨细胞样细胞增殖中发挥重要作用。刺激。较少的